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Survivin maintains the phosphorylation of Ser 62 in MYC. A , representative Western blot analysis of Survivin, MYC, phospho-MYC (P-MYC) Ser 62, and P-MYC Thr 58 levels in PANC-1 and MIA PaCa-2 cells treated with the indicated combinations of 25 μM chloroquine (CQ) and 250 nM YM155 for either 8 or 24 h. The relative changes in MYC phosphorylation at Ser 62 and Thr 58 were quantified using densitometry and listed below the corresponding blots. B , quantification of the relative changes in the phosphorylation of MYC at Ser 62 in cells treated without (DMSO) or with YM155 for 8 h. C , representative Western blot analysis of PP2A-A, PP2A-B, and PP2A-C expression in PANC-1 and MIA PaCa-2 cells treated without (DMSO) or with 250 nM YM155. D , representative Western blot analysis of Survivin and <t>CIP2A</t> expression in PANC-1 and MIA PaCa-2 cells treated with the indicated combinations of 25 μM chloroquine (CQ) and 250 nM YM155. E , representative Western blot analysis of CIP2A and MYC expression in PANC-1 cells expressing negative control (NC) and CIP2A-targeting shRNAs. F , representative Western blot analysis of Survivin and V5-tagged CIP2A expression in PANC-1 cells ectopically expressing V5-tagged CIP2A and treated with the indicated combinations of 25 μM chloroquine (CQ) and 250 nM YM155. G , quantitative PCR (qPCR) analysis of CIP2A mRNA levels, relative to actin mRNA levels, in PANC-1 and MIA PaCa-2 cells treated without (DMSO) or with 250 nM YM155 for 24 h. In ( A, C – F ), vinculin was used as the loading controls. All experiments involving Western blots were performed a minimum of three independent times, with similar results being obtained each time. In ( B and F ), data are means ± standard errors of at least three experiments; ∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by Student’s t test. PP2A, protein phosphatase 2A.
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Survivin maintains the phosphorylation of Ser 62 in MYC. A , representative Western blot analysis of Survivin, MYC, phospho-MYC (P-MYC) Ser 62, and P-MYC Thr 58 levels in PANC-1 and MIA PaCa-2 cells treated with the indicated combinations of 25 μM chloroquine (CQ) and 250 nM YM155 for either 8 or 24 h. The relative changes in MYC phosphorylation at Ser 62 and Thr 58 were quantified using densitometry and listed below the corresponding blots. B , quantification of the relative changes in the phosphorylation of MYC at Ser 62 in cells treated without (DMSO) or with YM155 for 8 h. C , representative Western blot analysis of PP2A-A, PP2A-B, and PP2A-C expression in PANC-1 and MIA PaCa-2 cells treated without (DMSO) or with 250 nM YM155. D , representative Western blot analysis of Survivin and CIP2A expression in PANC-1 and MIA PaCa-2 cells treated with the indicated combinations of 25 μM chloroquine (CQ) and 250 nM YM155. E , representative Western blot analysis of CIP2A and MYC expression in PANC-1 cells expressing negative control (NC) and CIP2A-targeting shRNAs. F , representative Western blot analysis of Survivin and V5-tagged CIP2A expression in PANC-1 cells ectopically expressing V5-tagged CIP2A and treated with the indicated combinations of 25 μM chloroquine (CQ) and 250 nM YM155. G , quantitative PCR (qPCR) analysis of CIP2A mRNA levels, relative to actin mRNA levels, in PANC-1 and MIA PaCa-2 cells treated without (DMSO) or with 250 nM YM155 for 24 h. In ( A, C – F ), vinculin was used as the loading controls. All experiments involving Western blots were performed a minimum of three independent times, with similar results being obtained each time. In ( B and F ), data are means ± standard errors of at least three experiments; ∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by Student’s t test. PP2A, protein phosphatase 2A.

Journal: The Journal of Biological Chemistry

Article Title: Oncogenic RAS promotes MYC protein stability by upregulating the expression of the inhibitor of apoptosis protein family member Survivin

doi: 10.1016/j.jbc.2022.102842

Figure Lengend Snippet: Survivin maintains the phosphorylation of Ser 62 in MYC. A , representative Western blot analysis of Survivin, MYC, phospho-MYC (P-MYC) Ser 62, and P-MYC Thr 58 levels in PANC-1 and MIA PaCa-2 cells treated with the indicated combinations of 25 μM chloroquine (CQ) and 250 nM YM155 for either 8 or 24 h. The relative changes in MYC phosphorylation at Ser 62 and Thr 58 were quantified using densitometry and listed below the corresponding blots. B , quantification of the relative changes in the phosphorylation of MYC at Ser 62 in cells treated without (DMSO) or with YM155 for 8 h. C , representative Western blot analysis of PP2A-A, PP2A-B, and PP2A-C expression in PANC-1 and MIA PaCa-2 cells treated without (DMSO) or with 250 nM YM155. D , representative Western blot analysis of Survivin and CIP2A expression in PANC-1 and MIA PaCa-2 cells treated with the indicated combinations of 25 μM chloroquine (CQ) and 250 nM YM155. E , representative Western blot analysis of CIP2A and MYC expression in PANC-1 cells expressing negative control (NC) and CIP2A-targeting shRNAs. F , representative Western blot analysis of Survivin and V5-tagged CIP2A expression in PANC-1 cells ectopically expressing V5-tagged CIP2A and treated with the indicated combinations of 25 μM chloroquine (CQ) and 250 nM YM155. G , quantitative PCR (qPCR) analysis of CIP2A mRNA levels, relative to actin mRNA levels, in PANC-1 and MIA PaCa-2 cells treated without (DMSO) or with 250 nM YM155 for 24 h. In ( A, C – F ), vinculin was used as the loading controls. All experiments involving Western blots were performed a minimum of three independent times, with similar results being obtained each time. In ( B and F ), data are means ± standard errors of at least three experiments; ∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by Student’s t test. PP2A, protein phosphatase 2A.

Article Snippet: The human PANC-1 and MIA PaCa-2 PDAC cell lines, as well as the MEFs, were grown in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum. pCDNA3 expression constructs encoding Myc-tagged Survivin (Sino Biological), HA-tagged MYC (Addgene #74164) , V5-tagged CIP2A (Addgene #119287) , GFP-tagged MYC (Addgene #42142) , and mCherry-LC3B (Addgene #40827) ( ) were transfected into MEFs using PEI (Sigma) and PDAC cells using FuGENE 6 (Promega) per the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Negative Control, Real-time Polymerase Chain Reaction

Diagram showing how KRAS-mediated upregulation of Survivin expression stabilizes MYC to promote PDAC cell growth. In normal cells, WT KRAS transiently activates ERK1/2, which in turn leads to the phosphorylation of Ser 62 (S62) in MYC, maintaining its stability. MYC is subsequently phosphorylated on Thr 58 (T58) by GSK-3β and dephosphorylated on Ser 62 by PP2A, which targets MYC for degradation (left side of diagram). However, mutant forms of KRAS (KRAS mt ) potently activate ERK1/2 and increase the phosphorylation of Ser 62 in MYC, enhancing its stability. We also found that mutant KRAS-induced ERK activation strongly upregulates Survivin expression, which further enhances MYC expression by both inhibiting its autophagy-dependent degradation, as well as by promoting the expression of CIP2A, preventing PP2A from dephosphorylating Ser 62 in MYC. These effects ensure that the proper amount of MYC is expressed in PDAC cells to support their optimal growth and to work together with KRAS to drive oncogenic transformation (right side of diagram). This figure was created using BioRender.com . ERK1/2, extracellular signal–regulated kinase 1/2; GSK-3β, glycogen synthase kinase 3β; PP2A, protein phosphatase 2A.

Journal: The Journal of Biological Chemistry

Article Title: Oncogenic RAS promotes MYC protein stability by upregulating the expression of the inhibitor of apoptosis protein family member Survivin

doi: 10.1016/j.jbc.2022.102842

Figure Lengend Snippet: Diagram showing how KRAS-mediated upregulation of Survivin expression stabilizes MYC to promote PDAC cell growth. In normal cells, WT KRAS transiently activates ERK1/2, which in turn leads to the phosphorylation of Ser 62 (S62) in MYC, maintaining its stability. MYC is subsequently phosphorylated on Thr 58 (T58) by GSK-3β and dephosphorylated on Ser 62 by PP2A, which targets MYC for degradation (left side of diagram). However, mutant forms of KRAS (KRAS mt ) potently activate ERK1/2 and increase the phosphorylation of Ser 62 in MYC, enhancing its stability. We also found that mutant KRAS-induced ERK activation strongly upregulates Survivin expression, which further enhances MYC expression by both inhibiting its autophagy-dependent degradation, as well as by promoting the expression of CIP2A, preventing PP2A from dephosphorylating Ser 62 in MYC. These effects ensure that the proper amount of MYC is expressed in PDAC cells to support their optimal growth and to work together with KRAS to drive oncogenic transformation (right side of diagram). This figure was created using BioRender.com . ERK1/2, extracellular signal–regulated kinase 1/2; GSK-3β, glycogen synthase kinase 3β; PP2A, protein phosphatase 2A.

Article Snippet: The human PANC-1 and MIA PaCa-2 PDAC cell lines, as well as the MEFs, were grown in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum. pCDNA3 expression constructs encoding Myc-tagged Survivin (Sino Biological), HA-tagged MYC (Addgene #74164) , V5-tagged CIP2A (Addgene #119287) , GFP-tagged MYC (Addgene #42142) , and mCherry-LC3B (Addgene #40827) ( ) were transfected into MEFs using PEI (Sigma) and PDAC cells using FuGENE 6 (Promega) per the manufacturer’s instructions.

Techniques: Expressing, Mutagenesis, Activation Assay, Transformation Assay